Oxidative stress alters renal D1 and AT1 receptor functions and increases blood pressure in old rats

Aging is associated with an increase in oxidative stress and blood pressure (BP). Renal dopamine D1 (D1R) and angiotensin II AT1 (AT1R) receptors maintain sodium homeostasis and BP. We hypothesized that age-associated increase in oxidative stress causes altered D1R and AT1R functions and high BP in aging. To test this, adult (3 mo) and old (21 mo) Fischer 344 × Brown Norway F1 rats were supplemented without/with antioxidant tempol followed by determining oxidative stress markers (urinary antioxidant capacity, proximal tubular NADPH-gp91phox, and plasma 8-isoprostane), D1R and AT1R functions, and BP. The D1R and AT1R functions were determined by measuring diuretic and natriuretic responses to D1R agonist (SKF-38393; 1 μg·kg(-1)·min(-1) iv) and AT1R antagonist (candesartan; 10 μg/kg iv), respectively. We found that the total urinary antioxidant capacity was lower in old rats, which increased with tempol treatment. In addition, tempol decreased the elevated NADPH-gp91phox and 8-isoprostane levels in old rats. Systolic, diastolic, and mean arterial BPs were higher in old rats and were reduced by tempol. Although SKF-38393 produced diuresis in both adult and old rats, urinary sodium excretion (UNaV) increased only in adult rats. While candesartan increased diuresis and UNaV in adult and old rats, the magnitude of response was greater in old rats. Tempol treatment in old rats reduced candesartan-induced increase in diuresis and UNaV. Our results demonstrate that diminished renal D1R and exaggerated AT1R functions are associated with high BP in old rats. Furthermore, oxidative stress may cause altered renal D1R and AT1R functions and high BP in old rats.     

Tempol improves vascular function in the mesenteric vascular bed of senescent rats

Ageing is associated with structural and functional alterations of the vasculature. The nature of age-related vascular disorders is not completely understood. Oxidative stress is hypothesized to play a crucial role in the pathophysiology of vascular complications. We investigated the effects of chronic treatment with the superoxide dismutase mimetic tempol (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl) on vascular function in the mesenteric vasculature of aged rats. Young (3 weeks) and old (40 weeks) Sprague-Dawley rats were treated with tempol (1 mM in drinking water) or vehicle for 3 weeks. Arterial blood pressure was slightly, but significantly, higher in old than in young rats. Tempol had no effect on arterial blood pressure. The vasoconstrictor responses to norepinephrine (NE) and serotonin (5-HT) were exaggerated in the mesenteric vascular bed (MVB) removed from old rats. Vasodilator responses to acetylcholine (ACh), papaverine (PPV), and isoprenaline (ISO) were reduced in the MVB of old rats in comparison with young rats. Chronic treatment of old rats with tempol normalized their responses to NE and 5-HT. The dilator responses to ACh, PPV, and ISO were similar between old rats receiving tempol and young rats. The present findings suggest that oxidative stress contributes to vascular dysfunction in the mesentery of old rats. The vasculoprotective effects of tempol remain to be elucidated.     

Mitochondrial Decay in the Brains of Old Rats: Ameliorating Effect of Alpha-Lipoic Acid and Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-carnitine

To investigate the mitochondrial decay and oxidative damage resulting from aging, the activities/kinetics of the mitochondrial complexes were examined in the brains of young and old rats as well as in old rats fed R-α-lipoic acid plus acetyl-l-carnitine (LA/ALC). The brain mitochondria of old rats, compared with young rats, had significantly decreased endogenous antioxidants and superoxide dismutase activity; more oxidative damage to lipids and proteins; and decreased activities of complex I, IV and V. Complex I showed a decrease in binding affinity (increase in K_m) for substrates. Feeding LA/ALC to old rats partially restored age-associated mitochondrial dysfunction to the levels of the young rats. These results indicate that oxidative mitochondrial decay plays an important role in brain aging and that a combination of nutrients targeting mitochondria, such as LA/ALC, could ameliorate mitochondrial decay through preventing mitochondrial oxidative damage. Special issue article in honor of Dr. Akitane Mori.     

Antioxidant properties of tea blunt ROS-dependent lipogenesis: beneficial effect on hepatic steatosis in a high fat-high sucrose diet NAFLD obese rat model

Oxidative stress could trigger lipid accumulation in liver and thus hepatic steatosis. Tea is able to prevent liver disorders, but a direct link between antioxidant capacities and prevention of steatosis has not been reported yet. We aimed to investigate such relationship in a rat model of high fat-high sucrose diet (HFS)-induced obesity and to explore more deeply the mechanisms in isolated hepatocytes. Wistar rats were divided into a control group (standard diet), an HFS group (high fat-sucrose diet) and an HFS + tea group (HFS diet with ad-libitum access to tea drink). Body weight, fat mass, glycemic parameters in blood, lipid and oxidative stress parameters in blood and liver were measured in each group after 14 weeks. Isolated hepatocytes were treated with the reactive oxygen species (ROS) inducer t-BHP in the presence or not of antioxidants (tempol or tea), and superoxide anion production and lipid accumulation were measured using specific fluorescent probes. We reported that the HFS diet highly increased hepatic lipids content, while tea consumption attenuated steatosis and improved the oxidative status (decrease in hepatic oxidative stress, increase in plasma total antioxidant capacity). The role of antioxidant properties of tea in such phenomenon was confirmed in primary cultured rat hepatocytes. Indeed, the increase of mitochondrial ROS production with t-BHP resulted in lipid accumulation in hepatocytes (positive linear regression), and antioxidants (tempol or tea) normalized both. We reported that the antioxidant properties of tea protect rats from an obesogenic HFS diet-induced hepatic steatosis by counteracting the ROS-dependent lipogenesis.     

Evidence for contribution of vascular NAD(P)H oxidase to increased oxidative stress in animal models of diabetes and obesity

It is well established that oxidative stress is enhanced in diabetes. However, the major in vivo source of oxidative stress is not clear. Here we show that vascular NAD(P)H oxidase may be a major source of oxidative stress in diabetic and obese models. In vivo electron spin resonance (ESR)/spin probe was used to evaluate systemic oxidative stress in vivo. The signal decay rate of the spin probe (spin clearance rate; SpCR) significantly increased in streptozotocin-induced diabetic rats 2 weeks after the onset of diabetes. This increase was completely normalized by treatment with the antioxidants alpha-tocopherol (40 mg/kg) and superoxide dismutase (5000 units/kg), and was significantly inhibited by treatment with a PKC-specific inhibitor, CGP41251 (50 mg/kg), and a NAD(P)H oxidase inhibitor, apocynin (5 mg/kg). Both obese ob/ob mice (10 weeks old) with mild hyperglycemia and Zucker fatty rats (11 weeks old) with normoglycemia exhibited significantly increased SpCR as compared with controls. Again, this increase was inhibited by treatment with both CGP41251 and apocynin. Oral administration of insulin sensitizer, pioglitazone (10 mg/kg), for 7 days also completely normalized SpCR values. These results suggest that vascular NAD(P)H oxidase may be a major source of increased oxidative stress in diabetes and obesity.     

Blood pressure has only minor influence on aldosterone-induced oxidative stress and DNA damage in vivo

Epidemiological studies found an increased kidney cancer risk in hypertensive patients. These patients frequently present an increase in the mineralocorticoid aldosterone (Ald) due to a stimulated renin angiotensin aldosterone system (RAAS). Recently, we showed pro-oxidative and genotoxic effects of Ald in vitro. Here, we investigated the influence of blood pressure on aldosterone-induced oxidative damage. To distinguish whether effects in Sprague–Dawley rats treated with Ald were caused by Ald or by increased blood pressure, the mineralocorticoid receptor (MR) antagonist spironolactone was administered in a subtherapeutical dose, not lowering the blood pressure, and hydralazine, a RAAS-independent vasodilator, was given to normalize the pressure. With the antioxidant tempol, oxidative stress-dependent effects were demonstrated. Ald treatment caused kidney damage and oxidative and nitrative stress. Structural DNA damage and the mutagenic oxidative base modification 7,8-dihydro-8-oxoguanine were increased, as well as DNA repair activity and nuclear NF-κB translocation. Spironolactone and tempol decreased all markers significantly, whereas hydralazine had just slight effects. These data comprise the first report of essentially blood pressure-independent tissue- and DNA-damaging effects of Ald. A fully activated MR and the production of reactive oxygen and nitrogen species were crucial for these effects.     

Abrogation of oxidative stress improves insulin sensitivity in the Ren-2 rat model of tissue angiotensin II overexpression

To evaluate the role of renin-angiotensin system (RAS)-mediated oxidative stress in insulin resistance (IR), we compared the effects of the angiotensin II (ANG II) receptor blocker (ARB) valsartan and a superoxide dismutase (SOD) mimetic, tempol, on whole body glucose tolerance and soleus muscle insulin-stimulated glucose uptake in transgenic hypertensive TG(mREN-2)27 (Ren-2) rats. Ren-2 rats and Sprague-Dawley (SD) controls were given valsartan (30 mg/kg) or tempol (1 mmol/l) in their drinking water for 21 days. IR was measured by glucose tolerance testing (1 g/kg glucose ip). IR index (AUC(glucose) x AUC(insulin)) was significantly higher in the Ren-2 animals compared with SD controls (30.5 +/- 7.0 x 10(6) arbitrary units in Ren-2 vs. 10.2 +/- 2.4 x 10(6) in SD, P < 0.01). Both valsartan and tempol treatment normalized Ren-2 IR index. Compared with SD controls (100%), there was a significant increase in superoxide anion production (measured by lucigenin-enhanced chemiluminescence) in soleus muscles of Ren-2 rats (133 +/- 15%). However, superoxide production was reduced in both valsartan- and tempol-treated (85 +/- 22% and 59 +/- 12%, respectively) Ren-2 rats. Insulin (INS)-mediated 2-deoxyglucose (2-DG) uptake (%SD basal levels) was substantially lower in Ren-2 rat soleus muscle compared with SD (Ren-2 + INS = 110 +/- 3% vs. SD + INS = 206 +/- 12%, P < 0.05). However, Ren-2 rats treated with valsartan or tempol exhibited a significant increase in insulin-mediated 2-DG uptake compared with untreated transgenic animals. Improvements in skeletal muscle insulin-dependent glucose uptake and whole body IR in rats overexpressing ANG II by ARB or SOD mimetic indicate that oxidative stress plays an important role in ANG II-mediated insulin resistance.     

Mineralocorticoid receptor activation: a major contributor to salt-induced renal injury and hypertension in young rats

Excessive salt intake is known to preferentially increase blood pressure (BP) and promote kidney damage in young, salt-sensitive hypertensive human and animal models. We have suggested that mineralocorticoid receptor (MR) activation plays a major role in kidney injury in young rats. BP and urinary protein were compared in young (3-wk-old) and adult (10-wk-old) uninephrectomized (UNx) Sprague-Dawley rats fed a high (8.0%)-salt diet for 4 wk. The effects of the MR blocker eplerenone on BP and renal injury were examined in the high-salt diet-fed young UNx rats. Renal expression of renin-angiotensin-aldosterone (RAA) system components and of inflammatory and oxidative stress markers was also measured. The effects of the angiotensin receptor blocker olmesartan with or without low-dose aldosterone infusion, the aldosterone synthase inhibitor FAD286, and the antioxidant tempol were also studied. Excessive salt intake induced greater hypertension and proteinuria in young rats than in adult rats. The kidneys of young salt-loaded rats showed marked histological injury, overexpression of RAA system components, and an increase in inflammatory and oxidative stress markers. These changes were markedly ameliorated by eplerenone treatment. Olmesartan also ameliorated salt-induced renal injury but failed to do so when combined with low-dose aldosterone infusion. FAD286 and tempol also markedly reduced urinary protein. UNx rats exposed to excessive salt at a young age showed severe hypertension and renal injury, likely primarily due to MR activation and secondarily due to angiotensin receptor activation, which may be mediated by inflammation and oxidative stress.     

Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-(alpha)-lipoic acid.

Oxidative stress has been implicated as a causal factor in the aging process of the heart and other tissues. To determine the extent of age-related myocardial oxidative stress, oxidant production, antioxidant status, and oxidative DNA damage were measured in hearts of young (2 months) and old (28 months) male Fischer 344 rats. Cardiac myocytes isolated from old rats showed a nearly threefold increase in the rate of oxidant production compared to young rats, as measured by the rates of 2,7-dichlorofluorescin diacetate oxidation. Determination of myocardial antioxidant status revealed a significant twofold decline in the levels of ascorbic acid (P = 0.03), but not alpha-tocopherol. A significant age-related increase (P = 0.05) in steady-state levels of oxidative DNA damage was observed, as monitored by 8-oxo-2'-deoxyguanosine levels. To investigate whether dietary supplementation with (R)-alpha-lipoic acid (LA) was effective at reducing oxidative stress, young and old rats were fed an AIN-93M diet with or without 0.2% (w/w) LA for 2 wk before death. Cardiac myocytes from old, LA-supplemented rats exhibited a markedly lower rate of oxidant production that was no longer significantly different from that in cells from unsupplemented, young rats. Lipoic acid supplementation also restored myocardial ascorbic acid levels and reduced oxidative DNA damage. Our data indicate that the aging rat heart is under increased mitochondrial-induced oxidative stress, which is significantly attenuated by lipoic acid supplementation.     

Lipoic acid and acetyl-carnitine reverse iron-induced oxidative stress in human fibroblasts

Iron overload occurs frequently in thalassemia and other disorders that require regular blood transfusions. Excess iron is toxic owing to the generation of free radicals that lead to oxidation of biomolecules and tissue damage. In order to identify compounds that reduce oxidative injury from iron, we evaluated alpha-lipoic acid (LA), a multifunctional antioxidant, in iron-overloaded primary human fibroblasts (IMR-90). Oxidant stress was measured using dichlorodihydrofluorescein diacetate that is converted to the fluorescent dichlorofluorescein (DCF) upon oxidation. Exposure to ferric ammonium citrate (FAC) increased the iron-content of IMR-90 cells and caused a rise in oxidant appearance. The addition of LA improved the cellular redox status and attenuated the iron-mediated rise in oxidants in a dose-dependent manner. The R- and RS-enantiomers of LA demonstrated similar antioxidant activity. N-tert-butyl hydroxylamine (NtBHA) treated cells also exhibited a decrease in DCF fluorescence, but at a much higher concentration compared with LA. The combination acetyl-L-carnitine (ALCAR) and LA exhibited superior antioxidant effect at all dose levels. We conclude that LA is highly effective in reversing oxidative stress arising from iron overload and that its antioxidant efficacy is further enhanced in combination with ALCAR.     

Dietary supplementation with (R)-alpha-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24-28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-alpha-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.     

Repeated Restraint Stress Induces Oxidative Damage in Rat Hippocampus

It has been shown that emotional stress may induce oxidative damage, and considerably change the balance between pro-oxidant and antioxidant factors in the brain. The aim of this study was to verify the effect of repeated restraint stress (RRS; 1 h/day during 40 days) on several parameters of oxidative stress in the hippocampus of adult Wistar rats. We evaluated the lipid peroxide levels (assessed by TBARS levels), the production of free radicals (evaluated by the DCF test), the total radical-trapping potential (TRAP) and the total antioxidant reactivity (TAR) levels, and antioxidant enzyme activities (SOD, GPx and CAT) in hippocampus of rats. The results showed that RRS induced an increase in TBARS levels and in GPx activity, while TAR was reduced. We concluded that RRS induces oxidative stress in the rat hippocampus, and that these alterations may contribute to the deleterious effects observed after prolonged stress.     

The effect of n-3 long-chain polyunsaturated fatty acids and lipoic acid on the heart in the ovariectomized rat model of menopause

Menopause occurs as consequence of ovarian senescence that leads to a drop of oestrogen hormone. The decreased oestrogen levels combined with the impairment of the redox system may contribute to the increased risk of postmenopausal cardiovascular disease. Supplementation with antioxidants may be an alternative to reduce cardiovascular risk. The study evaluated the effect of dietary supplementation with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and α-lipoic acid (LA) for a period of 16 weeks on oxidative stress biomarkers in the hearts of ovariectomized 3-month-old rats. Ovariectomy did not increase the level of the damage markers malondialdehyde and carbonyl, and both were decreased by LA supplementation. Ovariectomy increased the levels of the endogenous antioxidants glutathione, vitamin C and H₂O₂ consumption, after restoration by DHA, EPA, and LA supplementation. Vitamin E, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase are not altered by ovariectomy. Lipid and protein damage are not increased after ovariectomy and a portion of the endogenous antioxidants concomitantly increased, suggesting that hearts may be protected by these antioxidants. DHA, EPA, and LA restored these endogenous antioxidants, showing that all evaluated supplements are effective in modulating the antioxidant redox system in the heart. LA showed additional effect on redox markers, decreasing lipid and protein damage markers.     

Oxidative stress as regulatory factor for fatty-acid-induced uncoupling involving liver mitochondrial ADP/ATP and aspartate/glutamate antiporters of old rats

Palmitate-induced uncoupling, which involves ADP/ATP and aspartate/glutamate antiporters, has been studied in liver mitochondria of old rats (22-26 months) under conditions of lipid peroxidation and inhibition of oxidative stress by antioxidants--thiourea, Trolox, and ionol. It has been shown that in liver mitochondria of old rats in the absence of antioxidants and under conditions of overproduction of conjugated dienes, the protonophoric uncoupling activity of palmitate is not suppressed by either carboxyatractylate or aspartate used separately. However, the combination of carboxyatractylate and aspartate decreased uncoupling activity of palmitate by 81%. In this case, palmitate-induced uncoupling is limited by a stage insensitive to both carboxyatractylate and aspartate. In the presence of antioxidants, the palmitate-induced protonophoric uncoupling activity is suppressed by either carboxyatractylate or aspartate used separately. Under these conditions, palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter). In the absence of antioxidants, the uncoupling activity of palmitate is not suppressed by ADP either in the absence or in the presence of aspartate. However, in the presence of thiourea, Trolox, or ionol ADP decreased the uncoupling activity of palmitate by 38%. It is concluded that in liver mitochondria of old rats the development of oxidative stress in the presence of physiological substrates of ADP/ATP and aspartate/glutamate antiporters (ADP and aspartate) results in an increase of the protonophoric uncoupling activity of palmitate.     

Abnormalities of retinal metabolism in diabetes or experimental galactosemia. VI. Comparison of retinal and cerebral cortex metabolism,and effects of antioxidant therapy

Metabolic abnormalities observed in retina and in cerebral cortex were compared in diabetic rats and experimentally galactosemic rats. Diabetes or experimental galactosemia of 2 months duration significantly increased oxidative stress in retina, as shown by elevation of retinal thiobarbituric acid reactive substances (TBARS) and subnormal activities of antioxidant defense enzymes, but had no such effect in the cerebral cortex. Activities of sodium potassium adenosine triphosphatase [(Na,K)-ATPase] and calcium ATPase became subnormal in retina as well as in cerebral cortex. In contrast, protein kinase C (PKC) activity was elevated in retina but not in cerebral cortex in the same hyperglycemic rats. Dietary supplementation with an antioxidant mixture (containing ascorbic acid, Trolox, α-tocopherol acetate, N-acetyl cysteine, β-carotene, and selenium) prevented the diabetes- induced and galactosemia-induced elevation of retinal oxidative stress, the elevation of retinal PKC activity and the decrease of retinal ATPases. In cerebral cortex, administration of the antioxidant diet also prevented the diabetes-induced decreases in (Na,K)-ATPase and calcium ATPases, but had no effect on TBARS and activities of PKC and antioxidant-defense enzymes. The results indicate that retina and cerebral cortex differ distinctly in their response to elevation of tissue hexose, and that cerebral cortex is more resistant than retina to diabetes-induced oxidative stress. The greater resistance to oxidative stress in cerebral cortex, as compared to retina, is consistent with the resistance of cerebral cortex to microvascular disease in diabetes, and with a hypothesis that oxidative stress contributes to microvascular disease in diabetes. Dietary supplementation with these antioxidants offers a means to inhibit multiple hyperglycemia-induced retinal metabolic abnormalities.     

Acute antioxidant supplementation and skeletal muscle vascular conductance in aged rats: role of exercise and fiber type

Age-related increases in oxidative stress contribute to impaired skeletal muscle vascular control. However, recent evidence indicates that antioxidant treatment with tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) attenuates flow-mediated vasodilation in isolated arterioles from the highly oxidative soleus muscle of aged rats. Whether antioxidant treatment with tempol evokes similar responses in vivo at rest and during exercise in senescent individuals and whether this effect varies based on muscle fiber type composition are unknown. We tested the hypothesis that redox modulation via acute systemic tempol administration decreases vascular conductance (VC) primarily in oxidative hindlimb locomotor muscles at rest and during submaximal whole body exercise (treadmill running at 20 m/min, 5% grade) in aged rats. Eighteen old (25-26 mo) male Fischer 344 x Brown Norway rats were assigned to either rest (n = 8) or exercise (n = 10) groups. Regional VC was determined via radiolabeled microspheres before and after intra-arterial administration of tempol (302 μmol/kg). Tempol decreased mean arterial pressure significantly by 9% at rest and 16% during exercise. At rest, similar VC in 26 out of 28 individual hindlimb muscles or muscle parts following tempol administration compared with control resulted in unchanged total hindlimb muscle VC (control: 0.18 ± 0.02; tempol: 0.17 ± 0.05 ml·min(-1)·100 g(-1)·mmHg(-1); P > 0.05). During exercise, all individual hindlimb muscles or muscle parts irrespective of fiber type composition exhibited either an increase or no change in VC with tempol (i.e., ↑11 and ?17 muscles or muscle parts), such that total hindlimb VC increased by 25% (control: 0.93 ± 0.04; tempol: 1.15 ± 0.09 ml·min(-1)·100 g(-1)·mmHg(-1); P ≤ 0.05). These results demonstrate that acute systemic administration of the antioxidant tempol significantly impacts the control of regional vascular tone in vivo presumably via redox modulation and improves skeletal muscle vasodilation independently of fiber type composition during submaximal whole body exercise in aged rats.     

Influence of prenatal stress on the rat hypothalamic-pituitary-adrenal axis activity: the role of the brain glycocorticoid receptors

The effects of prenatal stress on the hypothalamic-pituitary-adrenal (HPA) axis activity and brain glycocorticoid receptors were studied in neonatal male and female offspring, as well as the influence of neonatal glycocorticoid receptors blockade on hormonal stress reactivity of adult rats. The results showed that there were sexual differences in plasma corticosterone level and corticosteroid binding in the cortex and hypothalamus of 5-day old control rats. Prenatal stress increased basal level of corticosterone in female rats, decreased corticosterone binding in hypothalamus and hippocampus of male and female rats, and increased corticosteroid receptor level in the male cortex. Neonatal administration of glycocorticoid receptor antagonist did not change plasma corticosterone level in 5-day old rats, but prolonged hormonal stress response of the HPA axis in adult male rats and increased hormonal stress response in female ones. The character of the IIPA axis activity of male and female rats with neonatal blockade of glycocorticoid receptors correspond to hormonal stress response of prenatal stressed rats. These data suggest that change of brain glycocorticoid receptors function in neonatal period of development might be one of the mechanisms of prenatal stress influence on the HPA axis activity in the adulthood.     

Standardization of a fluorometric assay for measuring oxidative stress in irradiated cells

The present study was undertaken to standardize a dichlorofluorescein (DCF) assay for measurement of radiation-induced oxidation of dichlorofluorescin (DCFH) substrate in MCF-10 cells. This assay was highly sensitive and capable of detecting increased DCFH oxidation in the cells exposed to gamma radiation at doses as low as 1.5 cGy with linear dose-response curves. However, the slope of the dose-response curves varied considerably from one experiment to another and was influenced by the fluorescent substrate concentration and cell density. To make the assay reproducible so that results obtained from different experiments could be compared, a series of conversion factors and equations have been established to normalize the data for these variables. The results demonstrate that the DCF assay, as standardized in the present study, is highly reproducible with acceptable assay precision. The normalized results can be compared from one experiment to another even when the experiments were performed using different fluorescent substrate concentrations and/or cell densities. Since changes in DCFH oxidation may be related to changes that are indicative of oxidative stress in cells, this assay can be useful to quantify radiation-induced oxidative stress and evaluate the efficacy of antioxidant agents in protection against radiation-induced oxidative stress.     

Dietary supplementation with (R)-α-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex

Abstract

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24–28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-α-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.